The research described in this application will concetrate on the use of primary neuronal cell cultures in the marine mollusk Aplysia as applied to two slow CNS processes--post-synaptic afterdischarge in the neuropeptidergic bag cells and the circadian oscillator in the eye of Aplysia. We will test the hypothesis that prolonged a afterdischage (approximately 30 minutes) is due to a rise in intracellular cAMP and that the membrane effects are mediated by protein phosphorylation catalyzed by activation of protein kinase. The basic membrane currents of the bag cell culture will be quantitated under voltage clamp and alteration of these currents by intracellular injection of the catalytic subunit of protein kinase will be investigated. Other investigations will concentrate on attempting to establish synaptic connections, in cell cultrue, between the pre-bag cell trigger neurons situated in the head ganglia and the bag cells. The nature of the transmitter, probably a peptide, that normally initiates afterdischarge in bag cells will be investigated by purifying peptides from the relevant head ganglion and assaying them on bag cells. The nature of the spontaneous circadian oscillator in the eye of Aplysia will be investigated by intra- and extra-cellular recordings from dissociated eye cells in cell culture. We will characterize light sensitivity in isolated photoreceptors (PRs) and PRs that are part of small groups of cells at different times of the day. Small clumps of cells, in cell culture, with a circadian light sensitivity will be examined by transmission electron microscopy of serial sections through the clump. Comparison of clumps with and without circadian oscillations should reveal, ideally, one cell type associated with the circadian rhythm. We will also use pulses of 3H-amino-acids followed by autoradiography to sample entire populations of cultured cells at different times of the day. We will determine whether circadian fluctuations in protein synthesis (known to occur in the intact eye) can be used to identify the circadian oscillator cell type in cell culture.